En Protein Forberedelse metode for High-throughput identifisering av proteiner samspill med et kjernefysisk kofaktor Bruke LC-MS / MS-analyse
Summary
Vi har etablert en metode for rensing av coregulatory interaksjons proteiner ved anvendelse av LC-MS / MS-system.
Abstract
Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.
Introduction
Protein-protein-interaksjoner spiller en viktig rolle i mange biologiske funksjoner. Som sådan, har disse interaksjoner vært innblandet i signaltransduksjon; protein transport over cellemembraner; celle metabolisme; og flere nukleære prosesser, blant annet DNA-replikasjon, DNA-skade reparasjon, rekombinasjon, og transkripsjon 1, 2, 3, 4. Identifisere proteiner som er involvert i disse interaksjonene er derfor avgjørende for å fremme vår forståelse av disse cellulære prosesser.
Immunoutfelling (IP) er et validert teknikk som brukes til å analysere protein-protein-interaksjoner. For å lette identifiseringen av ko-immunopresipitert proteiner, blir massespektrometri ofte benyttet 5, 6, 7, 8,9. Ved å målrette et kjent medlem av et proteinkompleks med et antistoff, er det mulig å isolere proteinkomplekset og deretter å identifisere de ukjente komponentene via massespektrometri-analyse. ARIP4 (androgen reseptor-samspill protein 4), en transkripsjons coregulator, samhandler med atom reseptor proteiner for å aktivere eller undertrykke sine mål arrangører i en kontekstavhengig måte 9, 10. For bedre å forstå de transkripsjonsregulerende mekanismer som styrer disse nukleære faktorer, beskriver vi en omfattende fremgangsmåte for å rense og identifisere ARIP4 samspill proteiner ved hjelp av LC-MS / MS-system.
Protocol
Representative Results
Discussion
Effektiv transfeksjon er avgjørende for å oppnå et vellykket resultat med denne protokoll. Derfor anbefaler vi at du bruker Western blot analyse for å fastslå de immunoutfelt FLAG-tagget protein nivåer. Dette trinnet gjør det mulig å verifisere at deres protein av interesse er riktig uttrykt, og dessuten at IP er vellykket gjennomført. FLAG-merket protein nivåer bør også sjekkes før massespektrometri analyse.
En mock prøven bør brukes som en negativ kontroll for å unngå å f…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.
Materials
| Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | 11668019 | |
| Protease Inhibitor Cocktail (EDTA free) (100x) | nacalai tesque | 03969-21 | |
| ANTI-FLAG M2 Affinity Gel | Sigma-Aldrich | A2220 | |
| Micro Bio-Spin Columns | BIO-RAD | 732-6204 |
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